WO2017007646A1 - Glycoprotéines d'enveloppe de variante c du vih-1 - Google Patents

Glycoprotéines d'enveloppe de variante c du vih-1 Download PDF

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WO2017007646A1
WO2017007646A1 PCT/US2016/039936 US2016039936W WO2017007646A1 WO 2017007646 A1 WO2017007646 A1 WO 2017007646A1 US 2016039936 W US2016039936 W US 2016039936W WO 2017007646 A1 WO2017007646 A1 WO 2017007646A1
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seq
hiv
vector
clade
antibodies
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PCT/US2016/039936
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English (en)
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Jayanta Bhattacharya
Suprit DESHPANDE
Shilpa PATIL
Rajesh Kumar
Bimal K. CHAKRABARTI
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International Aids Vaccine Initiative
Translational Health Science And Technology Institute
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Publication of WO2017007646A1 publication Critical patent/WO2017007646A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This application relates to novel HIV-1 envelope glycoproteins that may be utilized as an HIV-1 vaccine immunogen, as a native Env trimer mimic, for identification of small molecules, for use as an immunogen that binds specific HIV-1 broad neutralizing antibodies, for identification of small molecules for use as anti-viral compounds that bind specific HIV-1 envelope glycoprotein monomers and/or trimers, as antigens for crystallization and electron microscopy (EM) structural analysis and for the identification of broad neutralizing antibodies from HIV-1 infected individuals or vaccinated subjects or antibody or ligand libraries.
  • HIV-1 vaccine immunogen as a native Env trimer mimic
  • an immunogen that binds specific HIV-1 broad neutralizing antibodies for identification of small molecules for use as anti-viral compounds that bind specific HIV-1 envelope glycoprotein monomers and/or trimers
  • antigens for crystallization and electron microscopy (EM) structural analysis for the identification of broad neutralizing antibodies from HIV-1 infected individuals or vaccinated subjects or antibody or ligand libraries.
  • HIV human immunodeficiency virus
  • SIV simian immunodeficiency viruses
  • An infectious HIV particle consists of two identical strands of RNA, each approximately 9.2 kb long, packaged within a core of viral proteins. This core structure is surrounded by a phospholipid bilayer envelope derived from the host cell membrane that also includes virally-encoded membrane proteins (Abbas et al, Cellular and Molecular Immunology, 4th edition, W.B. Saunders Company, 2000, p. 454).
  • the HIV genome has the characteristic 5'-LTR-Gag-Pol-Env-LTR-3' organization of the retrovirus family. Long terminal repeats (LTRs) at each end of the viral genome serve as binding sites for transcriptional regulatory proteins from the host and regulate viral integration into the host genome, viral gene expression, and viral replication.
  • the HIV genome encodes several structural proteins.
  • the gag gene encodes structural proteins of the nucleocapsid core and matrix.
  • the pol gene encodes reverse transcriptase (RT), integrase (IN), and viral protease (PR) enzymes required for viral replication.
  • the tat gene encodes a protein that is required for elongation of viral transcripts.
  • the rev gene encodes a protein that promotes the nuclear export of incompletely spliced or unspliced viral RNAs.
  • the vz/gene product enhances the infectivity of viral particles.
  • the vpr gene product promotes the nuclear import of viral DNA and regulates G2 cell cycle arrest.
  • the vpu and nef genes encode proteins that down regulate host cell CD4 expression and enhance release of virus from infected cells.
  • the env gene encodes the viral envelope glycoprotein that is translated as a 160-kilodalton (kDa) precursor (gpl60) and cleaved by a cellular protease to yield the external 120-kDa envelope glycoprotein (gpl20) and the transmembrane 41-kDa envelope glycoprotein (gp41), which are required for the infection of cells (Abbas et al., Cellular and Molecular Immunology, 4th edition, W.B. Saunders Company, 2000, pp. 454-456).
  • kDa 160-kilodalton
  • gp41 transmembrane 41-kDa envelope glycoprotein
  • gpl40 is a modified form of the Env glycoprotein, which contains the external 120-kDa envelope glycoprotein portion and the extracellular part of the gp41 portion of Env and has characteristics of both gpl20 and gp41.
  • the nef gene is conserved among primate lentiviruses and is one of the first viral genes that is transcribed following infection. In vitro, several functions have been described, including down- regulation of CD4 and MHC class I surface expression, altered T-cell signaling and activation, and enhanced viral infectivity.
  • HIV infection initiates with viral particle binding on the cell membrane of target cells such as CD4 + T-cells, macrophages and dendritic cells.
  • target cells such as CD4 + T-cells, macrophages and dendritic cells.
  • HIV-1 uses a trimeric Env complex containing gpl20 and gp41 subunits (Burton et al, Nat Immunol. 2004 Mar;5(3):233-6).
  • the fusion potential of the Env complex is triggered by engagement of the CD4 receptor and a coreceptor, usually CCR5 or CXCR4.
  • the virus fuses with the target cell the RNA genome is reverse transcribed.
  • the resulting viral DNA integrates into the cellular genome, where it directs the production of new viral RNA, and thereby viral proteins and new virions.
  • virions bud from the infected cell membrane and establish productive infections in other cells. This process also kills the originally infected cell. HIV can also kill cells indirectly because the CD4 receptor on uninfected T- cells has a strong affinity for gpl20 expressed on the surface of infected cells. In this case, the uninfected cells bind, via the CD4 receptor-g l20 interaction, to infected cells and fuse to form a syncytium, which cannot survive. Destruction of CD4 + T-lymphocytes, which are critical to immune defense, is a major cause of the progressive immune dysfunction that is the hallmark of AIDS disease progression. The loss of CD4 + T cells seriously impairs the body's ability to fight most invaders, but it has a particularly severe impact on the defenses against viruses, fungi, parasites and certain bacteria, including mycobacteria.
  • HIV-1 envelope glycoprotein (Env) is the main viral protein involved in the entry of the virus and is also the primary target for neutralizing antibodies.
  • Neutralizing antibodies seem to work either by binding to the mature trimer on the virion surface and preventing initial receptor engagement events, or by binding after virion attachment and inhibiting the fusion process (Parren & Burton, Adv Immunol. 2001;77: 195-262). In the latter case, neutralizing antibodies may bind to epitopes whose exposure is enhanced or triggered by receptor binding.
  • it is not unexpected that HIV-1 has evolved multiple mechanisms to protect it from antibody binding (Johnson & Desrosiers, Annu Rev Med. 2002;53:499-518).
  • BCNAb response elicited during natural infection or through vaccination varies between host populations and has been shown to elicit a humoral immune response specific to infecting subtype and viral variants (Bandawe, G. P., et al. 2015. The Journal of infectious diseases 211 : 1461-1466; Dreja, H., et al. 2010. The Journal of general virology 91:2794-2803; Li, B., et al. 2006.
  • V1V2 including glycan moieties
  • glycan moieties Liao, H. X., et al. 2013. Immunity 38: 176-186; Pejchal, R, et al. 2011. Science 334: 1097-1103; Walker, L. M, et al. 2011. Nature 477:466-470; and Walker, L. M., et al. 2009. Science 326:285-289
  • gpl20-gp41 interface Blattner, C, et al. 2014. Immunity 40:669-680; and Scharf, L., et al. 2014.
  • HIV-1 clade C viruses mount distinct humoral immune responses in populations infected with not only genetically divergent clade C strains, but also due to differences in ethnicity and ancestral origin.
  • Some studies have shown genetic distinctness of Indian and non-Indian circulating HIV-1 clade C strains (Agnihorti, K., et al. 2004. AIDS Res. Hum. Retroviruses 20:889-894; Shankarappa, R., et al. 2001. Journal of virology 75:75: 10479-10487; and Shen, C, et al. 2011. PloS one 6:e25956).
  • Soluble BG505.664 Env displays a nearly native, cleaved, trimeric conformation; but requires stabilizing SOSIP mutations and co-expression of furin.
  • the present invention provides a vector comprising a nucleotide sequence encoding an HIV-1 clade C envelope glycoprotein the amino acid sequence of which has at least 95% identify with the amino acid sequence of SEQ ID No. 2 and a heterologous nucleic acid sequence.
  • the heterologous sequence may be a protein tag, regulatory sequence, or any sequence not naturally occurring with the HIV-1 clade C envelope glycoprotein.
  • the HIV-1 clade C envelope glycoprotein may be modified such that one or more amino acids located at a position within the VI region is substituted with an amino acid at a position corresponding to any of positions 126-159 of SEQ ID No. 2.
  • broad and cross clade neutralizing antibodies that target this specific region may be generated by further or new mutations in this region.
  • mutations occur in this region in response to antibodies and in order for a virus to become resistant to an immune response, the VI region would be an important epitope for generating broad and cross clade neutralizing antibodies.
  • the nucleotide sequence encoding an HIV-1 clade C envelope comprises SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, or SEQ ID No. 7.
  • the HIV-1 clade C envelope comprises the amino acid sequence of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, or SEQ ID No. 8.
  • the glycoproteins may bind broadly neutralizing antibodies isolated from HIV-1 infected individuals with high specificity in contrast to the binding of non-neutralizing antibodies.
  • the glycoproteins may be expressed on a cell surface or expressed in soluble form.
  • the present invention encompasses methods to express the glycoprotein in a soluble form and in a membrane bound form.
  • the transmembrane domain of the glycoprotein is removed to make the glycoprotein soluble.
  • the ability of the glycoprotein to bind with specificity to broadly neutralizing antibodies is dependent upon the protein maintaining its cleaved native trimeric state.
  • the native trimeric cleaved conformation of the HIV-1 clade C envelope glycoprotein may be maintained when the glycoprotein is expressed.
  • the glycoproteins may have specificity to the binding of broadly -neutralizing antibodies as compared to non-neutralizing antibodies.
  • the present invention relates to homologous glycoproteins that are modified, but share the characteristics of the glycoproteins of the present invention. Not being bound by a theory, small deviations from the specific sequences will result in a glycoprotein with the same characteristics.
  • the engineered or non- naturally occurring HIV-1 clade C envelope glycoprotein has at least 70%, 80%, 90%, preferably 95% identity to the protein of SEQ ID NO: 2, 4, 6 or 8, wherein the native trimeric cleaved conformation of gpl60 is maintained when the glycoprotein is expressed, and wherein the glycoprotein has specificity to the binding of broadly -neutralizing antibodies.
  • soluble envelope glycoproteins of the present invention have about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity to any of the sequences described.
  • the envelope glycoproteins have the characteristic of being cleaved in vivo and having a native conformation.
  • the cleaved envelope glycoproteins are preferentially bound by broadly neutralizing antibodies.
  • the nucleotide sequence is codon optimized.
  • the vector is configured to express the HIV-1 clade C envelope glycoproteins in a mammal.
  • the vector may be a plasmid.
  • the vector may be a virus.
  • the virus may be a lentivirus, adenovirus, adeno associated virus (AAV), or poxvirus.
  • AAV adeno associated virus
  • MVA is used as the viral vector.
  • the Clade C Env glycoproteins include stabilization mutations, wherein the stabilization mutations result in cysteine residues on either side of the cleavage site of gpl60, and wherein upon cleavage a disulfide bond can be formed.
  • Soluble, stabilized, proteolytically cleaved, trimeric gp41 proteins can be generated by engineering an intermolecular disulphide bond between gpl20 and gp41 (SOS), combined with a single residue change, I to P, within gp41 (SOSIP).
  • the nucleotide sequence encoding an HIV-1 clade C envelope glycoprotein may be operably linked to one or more expression control elements.
  • An isolated host cell line may comprise the vector.
  • the host cell line may be stably transformed with the vector.
  • the host cell line may be a prokaryotic host cell.
  • the present invention relates to a vaccine or immunogenic composition.
  • the vaccine or immunogenic composition preferably includes an excipient.
  • a genetic vaccine comprises a vector for expression of the envelope glycoprotein and a pharmaceutical excipient.
  • excipient is a natural or synthetic substance formulated alongside the active ingredient of a medication, included for the purpose of bulking-up formulations that contain potent active ingredients (thus often referred to as “bulking agents,” “fillers,” or “diluents”), or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption or solubility.
  • Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life. The selection of appropriate excipients also depends upon the route of administration and the dosage form, as well as the active ingredient and other factors.
  • the vaccine or immunogenic composition may include a vector that is a virus.
  • the virus may be a retrovirus, adenovirus, AAV, and a poxvirus.
  • MVA is used as the viral vector.
  • a vector may be introduced by transduction of a viral vector.
  • the present invention relates to a method of eliciting an immune response in a mammal comprising introducing the HIV-1 clade C envelope glycoproteins to the mammal.
  • the introducing may be by a viral vector.
  • the immune response can be elicited by use of a genetic vaccine.
  • An immune response may be elicited by introducing protein to the mammal.
  • the protein may be cleaved and solubilized before administration.
  • the protein may become cleaved after administration.
  • the protein may be introduced with an adjuvant.
  • the adjuvant may be a lecithin and may optionally be combined with an acrylic poly mer, a lecithin coated oil droplet in an oil-in-water emulsion or a lecithin and an acrylic polymer in an oil-in-water emulsion.
  • the adjuvant may be ISCOMATRIX or Adjuplex.
  • the adjuvant may comprise alum.
  • the present invention provides a genetic vaccine comprising a vector as described herein; and a pharmaceutical excipient.
  • the genetic vaccine may further comprise an adjuvant.
  • the present invention provides a method for generating an immune response in a subject comprising introducing into a subject an HIV-1 clade C envelope glycoprotein the amino acid sequence of which has at least 95% identity with the amino acid sequence of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6 or SEQ ID No. 8.
  • the vaccine or immunogenic composition may be a combination of the envelope glycoprotein of the present invention with another HIV antigen. This may include any number of HIV antigens encoded by the gag, pol, and env genes. Additionally, envelope glycoproteins from other strains of HIV can be used in the vaccine or immunogenic composition.
  • the vaccine may also include another vector that includes a nucleic acid encoding a clade B envelope glycoprotein.
  • the clade B envelope glycoprotein may be JRFL.
  • the vector or protein may be introduced ex vivo to at least one eukaryotic cell and the at least one eukaryotic cell may be introduced to the subject.
  • the eukaryotic cell may be derived from the subject.
  • the present invention provides an isolated non-naturally occurring nucleic acid molecule encoding an HIV-1 clade C envelope glycoprotein the amino acid sequence of which has at least 95% identity with the amino acid sequence of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6 or SEQ ID No. 8, wherein the nucleotide sequence is codon optimized.
  • the envelope glycoproteins of the present invention are used as a tool to determine other envelope glycoproteins that can bind to broadly neutralizing antibodies.
  • Crystal structures of antibodies bound to the envelope glycoproteins indicate epitopes important to bind broadly neutralizing antibodies.
  • structures of unknown envelope glycoproteins can be inferred based on the structure of the envelope glycoprotein of the present invention. In this way, envelope glycoproteins most likely to elicit broadly neutralized antibodies can be selected for further testing.
  • the envelope glycoprotein can be used to make therapeutic broadly neutralizing antibodies for treatment of patients in need thereof.
  • the envelope glycoproteins of the present invention are used to generate antibodies in an animal.
  • the animal expresses human light chain and heavy chain variable domain genes and is used to generate humanized antibodies.
  • the antibodies are administered to a patient in need thereof.
  • FIG. 1 Illustrates
  • FIG. 1 Illustrates an assessment of dependence of G37080 BCN antibodies to CD4 binding site (CD4bs) region of HIV-1 Env.
  • G37080 BCN plasma samples and VRCOl MAb concentration that neutralized 25711-2.4 by >80%
  • TripleMut core (A) and RSC3 (B) proteins were examined for their ability to neutralize 25711-2.4 Env pseudotyped virus in TZM-bl cell neutralization assay.
  • Figure 3 Illustrates binding of G37080 BCN plasma IgG to 4-2.J41 monomeric gpl20 (A) and BG505-SOSIP.664-D7324 cleaved trimenc gpl40 (B) soluble proteins were assessed by ELISA. IgG purified from HIV negative healthy donor and known MAbs were used as controls. Extent of binding of the depleted and undepleted G37080 BCN plasma with magnetic beads coated with 4-2.J41 monomeric gpl20 (C) and BG505-SOSIP.664 cleaved trimeric gpl40 to their respective proteins by ELISA (D).
  • Figure 4 Illustrates neutralization susceptibility of autologous Envs to contemporaneous G37080 BCN plasma and its follow up sample from the same donor.
  • Neutralization titers (median ID50) were obtained by titrating pseudotyped viruses expressing autologous Envs obtained from visit 1 and follow up G37080 plasma to contemporaneous plasma antibodies. Note that both the Envs obtained from follow up G37080 plasma (visit 2) were found to be resistant to contemporaneous autologous plasma, while Envs obtained from visit 1 G37080 plasma were found to be sensitive to follow up plasma antibodies.
  • FIG. 5 Illustrates mapping specificity of G37080 visit 2 BCN plasma antibodies using autologous Envs.
  • A Alignment of V1V2 amino acid sequences of sensitive and resistant autologous Envs obtained at both visits were done by using seqpublish available at HIV Los Alamos database (www.hiv.lanl.gov).
  • B Pseudoviruses expressing chimeric Envs prepared by swapping VI and V1V2 between sensitive (PG80vl .eJ7 and PG80vl .eJ19) and resistant (HVTR-PG80v2.eJ38) were tested to map specificity of G37080 BCN plasma by TZM-bl neutralization assay.
  • Figure 6 Illustrates a phylogenetic tree of the HIV-1 envelopes used to screen for neutralization breadth and potency of G37080 plasma antibodies. Neighbor-joining tree was constructed using Mega (version 5.2) software (scale bar of 0.5 amino acid substitution per site is highlighted). Hollow circles represent HIV-1 envelopes that were found to be resistant to neutralization by the G37080 plasma antibodies.
  • Figure 7. Illustrates the genetic relatedness of the autologous HIV-1 clade C envelope (gpl60) obtained from G37080 plasma. Maximum likelihood tree was prepared with 100 bootstrap replicates (scale bar of 0.005 amino acid substitution per site).
  • Figure 8 Illustrates the subtypes of the HIV-1 envelope clones obtained from the G37080 plasma. The subtyping is done by using a REGA HIV-1 subtyping tool - version 3.0 on Stanford University HIV Drug Resistance Database. Note that all the envelopes were found to belong to the subtype/clade C.
  • Figure 9 Illustrates
  • A Neutralization susceptibility of pseudoviruses expressing HIV-1 envelopes obtained from visit 1 and visit 2 G37080 plasma to autologous plasma antibodies obtained at visit 2.
  • Neutralization titers (ID50 values) for each virus were shown on top of the bar.
  • B V1V2 amino acid sequence alignment between the autologous envelopes obtained from G37080 plasma obtained at both visits. Potential N-linked glycan sites (PNGS) in VI are highlighted.
  • Figure 10 Illustrates neutralization susceptibility of pseudotyped viruses expressing autologous HIV-1 clade C Envs to the G37080 visit 2 plasma.
  • the neutralization ID50 values (reciprocal dilutions conferring 50% virus neutralization in TZM-bl cells) of each Envs are shown on top of each bar graph.
  • Figure 11 Illustrates an alignment of amino acid env sequences.
  • FIG. 12 Illustrates V3-C4 mediates neutralization escape of HVTR-PG80v2.e7
  • Env Note that a single P326I substitution in resistant Env (HVTR-PG80v2.eJ38) conferred it with increased susceptibility to contemporaneous autologous BCN plasma by >7.5 fold indicating this mutation played role in neutralization escape and resistance.)
  • Figure SI Illustrates the maximum likelihood phylogenetic tree constructed (with
  • Figure S2. Illustrates neutralization of HIV-1 Env pseudotyped viruses by BCN
  • Figure S3. Illustrates an assessment of the ability of G37080 BCN plasma antibodies in recognition of epitopes in gp41 MPER in CD4-unbound state.
  • Virus-antibody washout assay was carried out using an ultracentrifuge to remove unbound 10E8 MAb (A) and G37080 BCN plasma antibody (B) from the HIV-2/HIV-1 CIC chimeric virus.
  • Antibodies bound to HIV-2/HIV-1 CIC were incubated with TZM-bl cells and the degree of reduction in infection was assessed in a dose-dependent manner as described before. Note that while 10E8 MAb was able to recognize epitopes in MPER of HIV-2/HIV-1 C1C, G37080 BCN plasma failed to do so.
  • FIG. 1 Illustrates purification of codon optimized 4-2. J41 gpl20 monomeric protein.
  • A. 4-2. J41 gpl20 was codon optimized and sub cloned into pcDNA3.1Topo vector for efficient expression in 293T cells. A CD5 leader sequence and Kozak sequence were introduced upstream of the gpl20 sequence to facilitate efficient protein expression. Culture supernatants were purified in a Ni-NTA column and subsequently dialyzed to remove impurities.
  • B Purified gpl20 monomeric protein was run in a SDS-PAGE and stained with Coomassie blue stain.
  • Figure S5. Illustrates binding of 4-2.J41 monomeric gpl20 (A) and BG505- SOSIP.664 cleaved trimeric (B) soluble proteins coated on magnetic beads to neutralizing and non-neutralizing MAbs were assessed by flow cytometry. Beads coated with monomeric gpl20 and trimeric gpl40 were incubated with different MAbs and the extent of binding was detected by anti-human IgG coupled with FITC in a flow cytometer.
  • Figure S6 Illustrates neutralization of Env-pseudotyped viruses by undepleted and depleted PGT121 MAb (by BG505-SOSIP.664).
  • A Extent of depletion of PG121 MAb by repeat incubations of PGT121-BG505-SOSIP.664 soluble trimeric Env was monitored in a flow cytometer. Percent depletion of PGT121 MAb after second and tenth repeats are indicated in inset (pink). Note that following ten times depletion with BG505-SOSIP.664, only 65% depletion of PGT121 MAb was achieved.
  • B Extent of depletion of PG121 MAb by repeat incubations of PGT121-BG505-SOSIP.664 soluble trimeric Env was monitored in a flow cytometer. Percent depletion of PGT121 MAb after second and tenth repeats are indicated in inset (pink). Note that following ten times depletion with
  • Table 1 Illustrates neutralization breadth of Protocol G G37080 plasma samples collected at two different points tested against 57 panel Env-pseudotyped viruses.
  • Table 2 Illustrates examination of specificity of G37080 plasma antibodies obtained at both visits to HIV Env.
  • Table 3 Illustrates sensitivity of panel of Env-pseudotyped viruses by G37080 visit 2 BCN depleted with 4-2.J41 monomeric gpl20 and cleaved BG505-SOSIP.664 trimeric proteins in a TZM-bl neutralization assay.
  • the invention provides clade C HIV-1 Envelope glycoproteins isolated from an elite neutralizer whose plasma broadly and potently neutralizes greater than 90% of cross clade human immunodeficiency viruses.
  • Soluble, stabilized, proteolytically cleaved, trimeric proteins may be generated by engineering an intermolecular disulphide bond between gpl20 and gp41 (SOS), combined with a single residue change, I559P, within gp41 (SOSIP).
  • SOSIP gpl40 proteins based on the subtype A HIV-1 strain KNH1144 form particularly homogenous trimers compared to a prototypic strain (JR-FL, subtype B). Described in US Patent No.
  • 7,939,083 are the determinants of this enhanced stability which are located in the N-terminal region of KNH11144 gp41 and that, when substituted into heterologous Env sequences (e.g., JR-FL and Ba-L) they have a similarly beneficial effect on trimer stability.
  • These stabilized trimers retain the epitopes for several neutralizing antibodies and related agents (CD4-IgG2, bl2, 2G12, 2F5 and 4E10) and the CD4-IgG2 molecule, so that the overall antigenic structure of the gpl40 protein has not been adversely impaired by the trimer-stabilizing substitutions.
  • the protein is optimized for expression in mammals.
  • the Env protein can be used in a vaccine or immunogenic composition.
  • a DNA or genetic vaccine is used.
  • the vaccine includes a virus.
  • isolated Env protein is used as an immunogen.
  • the protein in another embodiment can be used as an antigen to produce broadly neutralizing antibodies in an animal.
  • the animal expresses human heavy and light chain variable regions to produce fully humanized antibodies. Such antibodies may be used as a therapeutic for HIV infected patients in need thereof.
  • the Env protein also can be used in screening for broadly neutralizing antibodies. Crystal structures of the envelope glycoprotein bound by broadly neutralizing antibodies can be used to determine important epitopes for envelope glycoproteins from other strains of HIV.
  • the present invention refers to various domains and sites present in all HIV-1 Env proteins. Such sequences and domains are well known in the art and can be found by accessing P04578-ENV_HV1H2 on the UniProt website (www.uniprot.org/uniprot P04578).
  • the Kennedy sequence refers to a hydrophilic sequence centered around position 740 of the gpl60 protein sequence and is C-termmal to the transmembrane or membrane spanning domain.
  • the transmembrane domain is generally located at position 685-705 in the gpl60 protein sequence.
  • the cleavage site is centered around position 512 of gpl60.
  • the MPER is located around position 662-683. Modification of any of these sites by mutation is understood to include modifications occurring to sites slightly deviating from these exact positions.
  • Env protein Env protein
  • Glycoprotein all refer to the envelope glycoprotein of HIV.
  • protein protein
  • peptide polypeptide
  • amino acid sequence amino acid sequence
  • the terms are used interchangeably herein to refer to polymers of amino acid residues of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.
  • the terms "antigen” or “immunogen” are used interchangeably to refer to a substance, typically a protein, which is capable of inducing an immune response in a subject.
  • the term also refers to proteins that are immunologically active in the sense that once administered to a subject (either directly or by administering to the subject a nucleotide sequence or vector that encodes the protein) is able to evoke an immune response of the humoral and/or cellular ty pe directed against that protein.
  • antibody includes intact molecules as well as fragments thereof, such as Fab, F(ab')2, Fv and scFv which are capable of binding the epitope determinant. These antibody fragments retain some ability to selectively bind with its antigen or receptor and include, for example:
  • Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
  • Fab' the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;
  • F(ab')2 the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction
  • F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds;
  • scFv including a genetically engineered fragment containing the variable region of a heavy and a light chain as a fused single chain molecule.
  • a "neutralizing antibody” may inhibit the entry of HIV-1 virus with a neutralization index >1.5 or >2.0. Broad and potent neutralizing antibodies may neutralize greater than about 50% of HIV-1 viruses (from diverse clades and different strains within a clade) in a neutralization assay. The inhibitory concentration of the monoclonal antibody may be less than about 25 mg/ml to neutralize about 50% of the input virus in the neutralization assay.
  • proteins including the antibodies and/or antigens of the invention may differ from the exact sequences illustrated and described herein.
  • the invention contemplates deletions, additions and substitutions to the sequences shown, so long as the sequences function in accordance with the methods of the invention.
  • particularly preferred substitutions are generally be conservative in nature, i.e., those substitutions that take place within a family of ammo acids.
  • amino acids are generally divided into four families: (1) acidic-aspartate and glutamate; (2) basic-lysine, arginine, histidine; (3) non-polar-alamne, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar-glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids.
  • nucleotide sequences and “nucleic acid sequences” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences, including, without limitation, messenger RNA (mRNA), DNA/RNA hybrids, or synthetic nucleic acids.
  • the nucleic acid can be single-stranded, or partially or completely double-stranded (duplex).
  • Duplex nucleic acids can be homoduplex or heteroduplex.
  • transgene may be used to refer to "recombinant" nucleotide sequences that may be derived from any of the nucleotide sequences encoding the proteins of the present invention.
  • the term “recombinant” means a nucleotide sequence that has been manipulated “by man” and which does not occur in nature, or is linked to another nucleotide sequence or found in a different arrangement in nature. It is understood that manipulated “by man” means manipulated by some artificial means, including by use of machines, codon optimization, restriction enzymes, etc.
  • the nucleotide sequences of the envelope glycoproteins may be codon optimized, for example the codons may be optimized for human use.
  • the nucleic acid molecules of the invention have a nucleotide sequence that encodes the antigens of the invention and can be designed to employ codons that are used in the genes of the subject in which the antigen is to be produced.
  • Many viruses, including HIV and other lentiviruses use a large number of rare codons and, by altering these codons to correspond to codons commonly used in the desired subject, enhanced expression of the antigens can be achieved.
  • the codons used are "humanized" codons, i.e., the codons are those that appear frequently in highly expressed human genes (Andre et al, J. Virol. 72: 1497-1503, 1998) instead of those codons that are frequently used by HIV.
  • Such codon usage provides for efficient expression of the transgenic HIV proteins in human cells. Any suitable method of codon optimization may be used. Such methods, and the selection of such methods, are well known to those of skill in the art.
  • there are several companies that will optimize codons of sequences such as Geneart (geneart.com).
  • Geneart geneart.com
  • the invention further encompasses nucleotide sequences encoding functionally and/or antigenically equivalent variants and derivatives of the envelope glycoproteins of the invention and functionally equivalent fragments thereof.
  • These functionally equivalent variants, derivatives, and fragments display the ability to retain antigenic activity. For instance, changes in a DNA sequence that do not change the encoded amino acid sequence, as well as those that result in conservative substitutions of amino acid residues, one or a few amino acid deletions or additions, and substitution of ammo acid residues by amino acid analogs are those which will not significantly affect properties of the encoded polypeptide.
  • Conservative amino acid substitutions are glycine/alanine; valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamic acid; serine/threonine/methionine; lysine/arginine; and phenylalanine/tyrosine/tryptophan.
  • the variants have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology or identity to the antigen, epitope, immunogen, peptide or polypeptide of interest.
  • the soluble envelope glycoproteins of the present invention have about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity to any of the sequences depicted in the figures and/or specification.
  • sequence identity or homology is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps.
  • sequence identity may be determined using any of a number of mathematical algorithms.
  • a nonlimiting example of a mathematical algorithm used for comparison of two sequences is the algorithm of Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1990; 87: 2264-2268, modified as in Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1993;90: 5873-5877.
  • Another example of a mathematical algorithm used for comparison of sequences is the algorithm of Myers & Miller, CABIOS 1988;4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Yet another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm as described in Pearson & Lipman, Proc. Natl. Acad. Sci. USA 1988; 85: 2444-2448.
  • WU-BLAST Woodington University BLAST
  • WU-BLAST version 2.0 executable programs for several UNIX platforms can be downloaded from ftp://blast.vvaistl.edu/blast/executables.
  • the nucleotide sequences of the present invention may be inserted into “vectors.”
  • vehicle is widely used and understood by those of skill in the art, and as used herein the term “vector” is used consistent with its meaning to those of skill in the art.
  • vector is commonly used by those skilled in the art to refer to a vehicle that allows or facilitates the transfer of nucleic acid molecules from one environment to another or that allows or facilitates the manipulation of a nucleic acid molecule.
  • any vector that allows expression of the envelope glycoprotein of the present invention may be used in accordance with the present invention.
  • the envelope glycoprotein of the present invention may be used in vitro (such as using cell-free expression systems) and/or in cultured cells grown in vitro in order to produce the encoded HIV envelope glycoprotein which may then be used for various applications such as in the production of proteinaceous vaccines.
  • any vector that allows expression of the envelope glycoprotein in vitro and/or in cultured cells may be used.
  • any vector that allows for the expression of the envelope glycoprotein of the present invention and is safe for use in vivo may be used.
  • the vectors used are safe for use in humans, mammals and/or laboratory animals.
  • the protein coding sequence should be "operably linked" to regulatory or nucleic acid control sequences that direct transcription and translation of the protein.
  • a coding sequence and a nucleic acid control sequence or promoter are said to be “operably linked” when they are covalently linked in such a way as to place the expression or transcription and/or translation of the coding sequence under the influence or control of the nucleic acid control sequence.
  • nucleic acid control sequence can be any nucleic acid element, such as, but not limited to promoters, enhancers, IRES, introns, and other elements described herein that direct the expression of a nucleic acid sequence or coding sequence that is operably linked thereto.
  • promoter will be used herein to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II and that when operationally linked to the protein coding sequences of the invention lead to the expression of the encoded protein.
  • the expression of the transgenes of the present invention can be under the control of a constitutive promoter or of an inducible promoter, which initiates transcription only when exposed to some particular external stimulus, such as, without limitation, antibiotics such as tetracycline, hormones such as ecdysone, or heavy metals.
  • the promoter can also be specific to a particular cell-type, tissue or organ.
  • suitable promoters and enhancers are known in the art, and any such suitable promoter or enhancer may be used for expression of the transgenes of the invention.
  • suitable promoters and/or enhancers can be selected from the Eukaryotic Promoter Database (EPDB).
  • the present invention relates to a recombinant vector expressing an envelope glycoprotein.
  • the present invention may encompass additional HIV antigens, epitopes or immunogens.
  • the additional HIV epitope is an HIV antigen, HIV epitope or an HIV immunogen, such as, but not limited to, the HIV antigens, HIV epitopes or HIV immunogens of U.S. Patent Nos. 7,341,731;
  • HIV, or immunogenic fragments thereof may be utilized as the HIV epitope.
  • any epitope recognized by an HIV antibody may be used in the present invention.
  • the anti-HIV antibodies of U.S. Patent Nos. 6,949,337, 6,900,010, 6,821,744, 6,768,004, 6,613,743, 6,534,312, 6,511,830, 6,489,131, 6,242,197, 6,114,143, 6,074,646, 6,063,564, 6,060,254, 5,919,457, 5,916,806, 5,871,732, 5,824,304, 5,773,247, 5,736,320, 5,637,455, 5,587,285, 5,514,541, 5,317,009, 4,983,529, 4,886,742, 4,870,003 and 4,795,739 are useful for the present invention.
  • the vectors used in accordance with the present invention should typically be chosen such that they contain a suitable gene regulatory region, such as a promoter or enhancer, such that the envelope glycoprotein of the invention can be expressed.
  • any suitable vector can be used depending on the application.
  • plasmids, viral vectors, bacterial vectors, protozoal vectors, insect vectors, baculovirus expression vectors, yeast vectors, mammalian cell vectors, and the like can be used.
  • Suitable vectors can be selected by the skilled artisan taking into consideration the characteristics of the vector and the requirements for expressing the envelope glycoprotein under the identified circumstances.
  • the aim is to express the envelope glycoprotein of the invention in vivo in a subject, for example in order to generate an immune response against an HIV-1 antigen and/or protective immunity against HIV-1
  • expression vectors that are suitable for expression on that subject, and that are safe for use in vivo, should be chosen.
  • any vectors that are suitable for such uses can be employed, and it is well within the capabilities of the skilled artisan to select a suitable vector.
  • the vectors used for these in vivo applications are attenuated to prevent the vector from amplifying in the subject.
  • plasmid vectors preferably they will lack an origin of replication that functions in the subject so as to enhance safety for in vivo use in the subject.
  • viral vectors preferably they are attenuated or replication-defective in the subject, again, so as to enhance safety for in vivo use in the subject.
  • An effective system for presenting HIV-1 Env as a membrane-anchored immunogen is through a genetic vaccine method such as a virus vector or plasmid DNA. Since it would be important for an HIV-1 Env vaccine candidate to bear structural resemblance and similar physio-chemical properties as the Env present on infectious viruses; expression of an efficiently cleaved Env in its native trimeric conformation on the cell surface through genetic vaccination, described herein, could prompt the immune system towards elicitation of potent neutralizing antibodies.
  • viral vectors are used.
  • Viral expression vectors are well known to those skilled in the art and include, for example, viruses such as adenoviruses, adeno-associated viruses (AAV), alphaviruses, herpesviruses, retroviruses and poxviruses, including avipox viruses, attenuated poxviruses, vaccinia viruses, and particularly, the modified vaccinia Ankara virus (MVA; ATCC Accession No. VR-1566).
  • viruses when used as expression vectors are innately non-pathogenic in the selected subjects such as humans or have been modified to render them non-pathogenic in the selected subjects.
  • replication-defective adenoviruses and alphaviruses are well known and can be used as gene delivery vectors.
  • the nucleotide sequences and/or envelope glycoproteins of the invention are administered in vivo, for example where the aim is to produce an immunogenic response in a subject.
  • a "subject" in the context of the present invention may be any animal.
  • the subject is a human, for example a human that is infected with, or is at risk of infection with, HIV-1.
  • the nucleotide sequences and/or envelope glycoproteins of the invention are preferably administered as a component of an immunogenic composition comprising the nucleotide sequences and'or envelope glycoproteins of the invention in admixture with a pharmaceutically acceptable carrier.
  • the immunogenic compositions of the invention are useful to stimulate an immune response against HIV-1 and may be used as one or more components of a prophylactic or therapeutic vaccine against HIV-1 for the prevention, amelioration or treatment of AIDS.
  • the nucleic acids and vectors of the invention are particularly useful for providing genetic vaccines, i.e. vaccines for delivering the nucleic acids encoding the envelope glycoproteins of the invention to a subject, such as a human, such that the envelope glycoproteins are then expressed in the subject to elicit an immune response.
  • compositions of the invention may be injectable suspensions, solutions, sprays, lyophilized powders, syrups, elixirs and the like. Any suitable form of composition may be used.
  • a nucleic acid or vector of the invention having the desired degree of purity, is mixed with one or more pharmaceutically acceptable carriers and/or excipients.
  • the carriers and excipients must be "acceptable" in the sense of being compatible with the other ingredients of the composition.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, or combinations thereof, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzvl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobul
  • An immunogenic or immunological composition can also be formulated in the form of an oil-in-water emulsion.
  • the oil-in-water emulsion can be based, for example, on light liquid paraffin oil (European Pharmacopea type); isoprenoid oil such as squalane, squalene, EICOSANETM or tetratetracontane; oil resulting from the oligomerization of alkene(s), e.g., isobutene or decene; esters of acids or of alcohols containing a linear alkyl group, such as plant oils, ethyl oleate, propylene glycol di(caprylate/caprate), glyceryl trif caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, e.g., isostearic acid esters.
  • the oil advantageously is used in combination with emulsifiers to form the emulsion.
  • the emulsifiers can be nonionic surfactants, such as esters of sorbitan, mannide (e.g., anhydromannitol oleate), glycerol, polyglycerol, propylene glycol, and oleic, isostearic, ricinoleic, or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, such as the Pluronic® products, e.g., L121.
  • the adjuvant can be a mixture of emulsifier(s), micelle-forming agent, and oil such as that which is commercially available under the name Provax® (IDEC Pharmaceuticals, San Diego, CA).
  • the immunogenic compositions of the invention can contain additional substances, such as wetting or emulsifying agents, buffering agents, or adjuvants to enhance the effectiveness of the vaccines (Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, (ed.) 1980).
  • Adjuvants may also be included.
  • Adjuvants include, but are not limited to, mineral salts (e.g., A1K(S04)2, AlNa(S04)2, A1NH(S04)2, silica, alum, Al(OH)3, Ca3(P04)2, kaolin, or carbon), polynucleotides with or without immune stimulating complexes (ISCOMs) (e.g., CpG oligonucleotides, such as those described in Chuang, T.H. et al, (2002) J. Leuk. Biol. 71(3): 538- 44; Ahmad-Nejad, P. et al (2002) Eur. J. Immunol.
  • mineral salts e.g., A1K(S04)2, AlNa(S04)2, A1NH(S04)2, silica, alum, Al(OH)3, Ca3(P04)2, kaolin, or carbon
  • ISCOMs immune stimulating complexes
  • saponins such as QS21, QS17, and QS7 (U.S. Patent Nos. 5,057,540; 5,650,398; 6,524,584; 6,645,495), monophosphoryl lipid A, in particular, 3-de-O- acylated monophosphoryl lipid A (3D-MPL), imiquimod (also known in the art as IQM and commercially available as Aldara®; U.S. Patent Nos. 4,689,338; 5,238,944; Zuber, A.K. et al (2004) 22(13-14): 1791-8), and the CCR5 inhibitor CMPD167 (see Veazey, R.S. et al (2003) J. Exp. Med. 198: 1551-1562).
  • Aluminum hydroxide or phosphate are commonly used at 0.05 to 0.1% solution in phosphate buffered saline.
  • cytokines such as, but not limited to, IL-2, IL-4, GM-CSF, IL-12, IL-15 IGF-1, IFN-a, IFN- ⁇ , and IFN- ⁇
  • immunoregulatory proteins such as CD40L (ADX40; see, for example, WO03/063899)
  • CD la ligand of natural killer cells also known as CRONY or a-galactosyl ceramide; see Green, T.D. et al, (2003) J. Virol.
  • immunostimulatory fusion proteins such as IL-2 fused to the Fc fragment of immunoglobulins (Barouch et al, Science 290:486- 492, 2000) and co-stimulatory molecules B7.1 and B7.2 (Boyer), all of which can be administered either as proteins or in the form of DNA, on the same expression vectors as those encoding the antigens of the invention or on separate expression vectors.
  • the adjuvants may be lecithin combined with an acrylic polymer (Adjuplex-LAP), lecithin coated oil droplets in an oil-in- water emulsion (Adjuplex-LE) or lecithin and acrylic polymer in an oil-in-water emulsion (Adjuplex-LAO) (Advanced BioAdjuvants (ABA)).
  • Adjuplex-LAP acrylic polymer
  • Adjuplex-LE lecithin coated oil droplets in an oil-in- water emulsion
  • Adjuplex-LAO Advanced BioAdjuvants
  • the immunogenic compositions can be designed to introduce the nucleic acids or expression vectors to a desired site of action and release it at an appropriate and controllable rate.
  • Methods of preparing controlled-release formulations are known in the art.
  • controlled release preparations can be produced by the use of polymers to complex or absorb the immunogen and/or immunogenic composition.
  • a controlled-release formulation can be prepared using appropriate macromolecules (for example, polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) known to provide the desired controlled release characteristics or release profile.
  • Another possible method to control the duration of action by a controlled-release preparation is to incorporate the active ingredients into particles of a polymeric material such as, for example, polyesters, polyamino acids, hydrogels, polylactic acid, polyglycolic acid, copolymers of these acids, or ethylene vinylacetate copolymers.
  • a polymeric material such as, for example, polyesters, polyamino acids, hydrogels, polylactic acid, polyglycolic acid, copolymers of these acids, or ethylene vinylacetate copolymers.
  • microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacrylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • Suitable dosages of the nucleic acids and expression vectors of the invention in the immunogenic composition of the invention can be readily determined by those of skill in the art.
  • the dosage of the immunogens can vary depending on the route of administration and the size of the subject.
  • Suitable doses can be determined by those of skill in the art, for example by measuring the immune response of a subject, such as a laboratory animal, using conventional immunological techniques, and adjusting the dosages as appropriate.
  • Such techniques for measuring the immune response of the subject include but are not limited to, chromium release assays, tetramer binding assays, IFN- ⁇ ELISPOT assays, IL-2 ELISPOT assays, intracellular cytokine assays, and other immunological detection assays, e.g., as detailed in the text "Antibodies: A Laboratory Manual” by Ed Harlow and David Lane.
  • the immunogenic compositions of the invention are ideally administered to a subject in advance of HIV infection, or evidence of HIV infection, or in advance of any symptom due to AIDS, especially in high-risk subjects.
  • the prophylactic administration of the immunogenic compositions can serve to provide protective immunity of a subject against HIV-1 infection or to prevent or attenuate the progression of AIDS in a subject already infected with HIV-1.
  • the immunogenic compositions can serve to ameliorate and treat AIDS symptoms and are advantageously used as soon after infection as possible, preferably before appearance of any symptoms of AIDS but may also be used at (or after) the onset of the disease symptoms.
  • the immunogenic compositions can be administered using any suitable delivery method including, but not limited to, intramuscular, intravenous, intradermal, mucosal, and topical delivery. Such techniques are well known to those of skill in the art. More specific examples of delivery methods are intramuscular injection, intradermal injection, and subcutaneous injection. However, delivery need not be limited to injection methods. Further, delivery of DNA to animal tissue has been achieved by cationic liposomes (Watanabe et al, (1994) Mol. Reprod. Dev.
  • delivery routes can be oral, intranasal or by any other suitable route. Delivery also be accomplished via a mucosal surface such as the anal, vaginal or oral mucosa.
  • Immunization schedules are well known for animals (including humans) and can be readily determined for the particular subject and immunogenic composition.
  • the immunogens can be administered one or more times to the subject.
  • there is a set time interval between separate administrations of the immunogenic composition typically it ranges from 10 days to several weeks, and is often 2, 4, 6 or 8 weeks.
  • the interval is typically from 2 to 6 weeks.
  • the immunization regimes typically have from 1 to 6 administrations of the immunogenic composition, but may have as few as one or two or four.
  • the methods of inducing an immune response can also include administration of an adjuvant with the immunogens. In some instances, annual, biannual or other long interval (5-10 years) booster immunization can supplement the initial immunization protocol.
  • the present methods also include a variety of prime-boost regimens, for example DNA prime-Adenovirus boost regimens.
  • one or more priming immunizations are followed by one or more boosting immunizations.
  • the actual immunogenic composition can be the same or different for each immunization and the type of immunogenic composition (e.g., containing protein or expression vector), the route, and formulation of the immunogens can also be varied.
  • an expression vector is used for the priming and boosting steps, it can either be of the same or different type (e.g., DNA or bacterial or viral expression vector).
  • Prime-boost regimen provides for two priming immunizations, four weeks apart, followed by two boosting immunizations at 4 and 8 weeks after the last priming immunization. It should also be readily apparent to one of skill in the art that there are several permutations and combinations that are encompassed using the DNA, bacterial and viral expression vectors of the invention to provide priming and boosting regimens.
  • a specific embodiment of the invention provides methods of inducing an immune response against HIV in a subject by administering an immunogenic composition of the invention, preferably comprising an adenovirus vector containing DNA encoding one or more of the epitopes of the invention, one or more times to a subject wherein the epitopes are expressed at a level sufficient to induce a specific immune response in the subject.
  • an immunogenic composition of the invention preferably comprising an adenovirus vector containing DNA encoding one or more of the epitopes of the invention, one or more times to a subject wherein the epitopes are expressed at a level sufficient to induce a specific immune response in the subject.
  • Such immunizations can be repeated multiple times at time intervals of at least 2, 4 or 6 weeks (or more) in accordance with a desired immunization regime.
  • the immunogenic compositions of the invention can be administered alone, or can be co-administered, or sequentially administered, with other HIV immunogens and/or HIV immunogenic compositions, e.g., with "other" immunological, antigenic or vaccine or therapeutic compositions thereby providing multivalent or "cocktail” or combination compositions of the invention and methods of employing them.
  • the ingredients and manner (sequential or co-administration) of administration, as well as dosages can be determined taking into consideration such factors as the age, sex, weight, species and condition of the particular subject, and the route of administration.
  • the other HIV immunogens can be administered at the same time or at different times as part of an overall immunization regime, e.g., as part of a prime-boost regimen or other immunization protocol.
  • HIVA (described in WO 01/47955), which can be administered as a protein, on a plasmid (e.g., pTHr.HIVA) or in a viral vector (e.g., MVA.HIVA).
  • RENTA (described in PCT/US2004/037699), which can also be administered as a protein, on a plasmid (e.g., pTHr.RENTA) or in a viral vector (e.g., MVA.RENTA).
  • one method of inducing an immune response against HIV in a human subject comprises administering at least one priming dose of an HIV immunogen and at least one boosting dose of an HIV immunogen, wherein the immunogen in each dose can be the same or different, provided that at least one of the immunogens is an epitope of the present invention, a nucleic acid encoding an epitope of the invention or an expression vector, preferably a VSV vector, encoding an epitope of the invention, and wherein the immunogens are administered in an amount or expressed at a level sufficient to induce an HIV-specific immune response in the subject.
  • the HIV-specific immune response can include an HIV- specific T-cell immune response or an HIV-specific B-cell immune response.
  • Such immunizations can be done at intervals, preferably of at least 2-6 or more weeks.
  • Pseudotyped viruses may be generated by co-transfecting cells with at least two plasmids encoding the soluble Env cDNA of the present invention and the rest of the HIV genome separately.
  • the Env gene may be replaced by the firefly luciferase gene.
  • Transfectant supernatants containing pseudotyped virus may be co-incubated overnight with B cell supernatants derived from activation of an infected donor's primary peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • Cells stably transfected with and expressing CD4 plus the CCR5 and CXCR4 coreceptors may be added to the mixture and incubated for 3 days at 37° C. Infected cells may be quantified by luminometry.
  • the soluble envelope glycoproteins of the present invention may be crystallized in combination with PG9, VRCOl, PGT121, 10E8, PGT145 or PG16 or with any other neutralizing antibodies, including those described in the examples, to determine the exact molecular surface where the soluble envelope glycoprotein binds with the neutralizing antibody to design HIV-1 immunogens.
  • Crystals of the invention may be obtained by conventional means as are well- known in the art of protein crystallography, including batch, liquid bridge, dialysis, vapor diffusion and hanging drop methods (see, e.g., Johnson et al., Biochemistry. 1982 Sep 28;21(20):4839-43; Brayer & McPherson, J Biol Chem. 1982 Apr 10;257(7):3359-61; McPherson &Weickmann, J Biomol Struct Dyn. 1990 Apr;7(5): 1053-60; and Koszelak et al, J Mol Biol. 1989 Sep 20;209(2):323-5; Weber et al., Acta Crystallogr B. 1991 Feb 1;47 ( Pt 1): 116-27 and Weber, Methods Enzymol. 1991;202:727-41).
  • the crystals of the invention are grown by dissolving a substantially pure neutralizing antibody, such as PG9, VRCOl, PGT121, 10E8, PGT145 or PG16, and soluble envelope glycoprotein in an aqueous buffer containing a precipitant at a concentration just below that necessary to precipitate the protein. Water is removed by controlled evaporation to produce precipitating conditions, which are maintained until crystal growth ceases.
  • a substantially pure neutralizing antibody such as PG9, VRCOl, PGT121, 10E8, PGT145 or PG16
  • the crystals of the invention and particularly the atomic structure co-ordinates obtained therefrom, have a wide variety of uses.
  • the crystals and structure co-ordinates are particularly useful for identifying protein domains that bind to a neutralizing antibody, such as PG9, VRCOl, PGT121, 10E8, PGT145 or PG16, and thus are useful to elicit anti-HIV antibodies.
  • a neutralizing antibody such as PG9, VRCOl, PGT121, 10E8, PGT145 or PG16
  • Such protein domains may be useful in eliciting clade C anti-HIV antibodies, however variants may be useful in eliciting clade A, B, D or E anti-HIV antibodies.
  • the structure co-ordinates may be used as phasing models in determining the crystal structures of a synthetic or mutated neutralizing antibody, such as PG9, VRCOl, PGT121, 10E8, PGT145 or PG16, domains, as well as the structures of co-crystals of such domains with ligands.
  • a synthetic or mutated neutralizing antibody such as PG9, VRCOl, PGT121, 10E8, PGT145 or PG16
  • the method may use the co-ordinates of atoms of interest of a neutralizing antibody which are in the vicinity of the binding region in order to model the pocket in which the ligand binds. These coordinates may be used to define a space which is then screened "in silico" against a candidate molecule.
  • the invention provides a computer-based method of rational drug or compound design or identification which comprises: providing the coordinates of at least selected co-ordinates; providing the structure of a candidate compound; and fitting the structure of the candidate to the selected co-ordinates.
  • a neutralizing antibody such as PG9, VRCOl, PGT121, 10E8, PGT145 or PG16, as defined by its co-ordinates which represent the active site or binding region.
  • a neutralizing antibody such as PG9, VRCOl, PGT121, 10E8, PGT145 or PG16
  • the present invention provides advantages relating to development of new treatments and research tools based on the discovery of envelope ⁇ env) genes obtained from a very broad and potent cross clade neutralizing plasma (G37080).
  • the plasma has shown unique antibody specificities not identified before.
  • the G37080 plasma neutralizing antibodies were found to bind to epitopes on cleaved trimenc HIV-1 envelope protein.
  • HVTR-PG80vl.eJ7 and HVTR-PG80vl.eJ19 and resistant (HVTR- PG80v2.eJ38) autologous HIV-1 envelopes
  • Applicants found antibody specificities in the VI region, targeting epitopes not reported before.
  • HIV-1 clade C envelopes are an improved platform towards design of novel HIV-1 envelope based immunogen/s in that if they are able to elicit similar antibody responses upon immunization, it would be able to neutralize a wide range of HIV-1 strains circulating globally.
  • HIV-1 envelope based immunogens that have been taken forward into advanced stages (for e.g., BG505, JRFL) have not originated from broadly neutralizing plasma.
  • envelope clones Hoffenberg, S., et al. 2013.
  • HVTR- PG80vl.eJ7 and HVTR-PG80vl.eJ19 envelopes were found to be sensitive and displayed novel epitopes in the VI loop targeted by the broadly neutralizing G37080 plasma antibodies making these envelopes attractable for use as a platform for immunogen design and vaccine studies.
  • these isolated envelope clones may be used to design and develop HIV-1 envelope based immunogen/s and may also be used in drug discovery in addition to vaccine discovery that interferes at the virus entry level. These envelope clones will be very useful for screening serum and monoclonal neutralizing antibodies both at industrial and academic levels.
  • Applicants examined plasma samples obtained from two hundred asymptomatic and anti-retroviral therapy (ART) naive Indian donors, who at the time of blood collection were chronically infected with HIV-1, for the presence of cross neutralizing plasma antibodies. Applicants deciphered the molecular specificities of plasma antibodies obtained from a clade C infected elite neutralizer, who showed exceptional neutralization breadth across clades of different geographical origins by recognizing epitopes in V1V2 region, a region previously not reported to be recognized on cleaved trimeric Env.
  • ART anti-retroviral therapy
  • the present study under IAVI Protocol G was designed to (i) screen for identification of plasma antibodies obtained from chronically infected Indian donors with HIV-1 clade C with substantial breadth towards neutralizing cross clade HIV-1 primary variants and (ii) to elucidate their molecular specificities associated with neutralization breadth. Due to the genetic heterogeneity of the protein sequences of the HIV-1 clade C Envs circulating strains in India compared to that of non-Indian clade C circulating strains, particularly in the African continent ( Figure S I), as well due to likely differences in host genetics associated with modulation of humoral immune responses between populations with differences in their ancestral origin, Applicants predicted that the specificities of antibodies developed in vivo associated with neutralization breadth and potency would be different.
  • G37080 BCN plasma was found to potently neutralize pseudoviruses expressing Indian clade C Env.
  • the neutralization score of the G37080 BCN plasma was found to be 2.5 (Simek, M. D., et al. 2009. Journal of virology 83:7337-7348).
  • Neutralization sensitivity of Env-pseudotyped viruses was found to be correlated with the serum IgG ( Figure S2), suggesting broad neutralization was associated with IgG-specific response.
  • gpl60 HIV-1 clade C full length envelope genes from a slow progressing Indian patient who plasma showed exceptional neutralization breadth and potency across different clades of distinct geographical origin
  • the gp!60 clones were amplified from two time points and were designated as 'Visit 1" and 'Visit 2'.
  • These gp!60 genes were cloned into pcDNA3.1/V5-His-TOPO vector and are capable of producing functional envelope- pseudotyped viruses and were subsequently used in neutralizing antibody assays.
  • the names and description of these gp!60 are given in Table A.
  • Applicants prepared chimeric Envs between a sensitive (HVTR-PG80vl .eJ7 and HVTR-PG80vl.eJ19) and the resistant (HVTR-PG80v2.eJ38) autologous Envs by first swapping the V1V2 regions as their amino acid sequences differed maximally in this region ( Figure 5A) and since other regions of the envelope were not found to contribute in modulating sensitivity to autologous plasma antibodies.
  • Applicants characterized the molecular specificity of plasma antibodies obtained from an Indian elite neutralizer infected with HIV-1 clade C that showed exceptional cross neutralization of different clades of distinct geographical origins.
  • G37080 plasma was found to be the broadest and most potent neutralizing plasma amongst 200 plasma screened against 54 Env-pseudoty ped viruses of distinct subtypes and origins. Both plasma samples collected from the G37080 donor at different time points eight months apart showed similar neutralization breadth with modest increase in median ID50 value, indicating association of sustained maturation of antibody producing B cells in this individual.
  • the G37080 plasma antibodies did not show dependence on the N160/K169 and N332 epitopes in V2 and V3 regions respectively; nonetheless Applicants found evidence that the G37080 BCN antibodies possibly target complex sugar moieties as N- acetylglucosaminyltransferase I (GnTI)-deficient HEK293S derived Envs displayed significant reduction in susceptibility to the G37080 BCN antibodies. This observation is in contrast to the findings of Doores and Burton (Doores, K. J., and D. R. Burton. 2010.
  • Wibmer et al Wibmer, C. K , et al. 2013. PLoS pathogens 9:el003738) recently demonstrated the association of evolution of a broadly neutralizing antibody response in a clade C infected donor with escape variants and shifts in antibody specificities from recognition of epitopes in V2 to the CD4bs.
  • the G37080 neutralizing plasma antibodies obtained from both the visits with 8 months apart were found not to be absorbed out by the TripleMut (Chakrabarti, B. K., et al. 2013. Journal of virology 87: 13239- 13251; and Feng, Y., et al. 2012.
  • G37080 BCN antibodies As described herein, Applicants eliminated the likelihood of use of residues in V1V2 loop by G37080 BCN antibodies as epitopes that are targets of the known broad and potent neutralizing monoclonal antibodies; thus G37080 BCN antibodies appear to target novel contact points in V1V2 as epitopes.
  • variable regions within the HIV-1 gpl20 has been demonstrated to contain epitopes targeted by the autologous as well as BCN antibodies in different studies (Chaillon, A., et al. 2011. Journal of virology 85:3642-3648; Doria-Rose, N. A, et al. 2012. Journal of virology 86:8319-8323; Doria-Rose, N. A., et al. 2014. Nature 509:55-62; Harrington, P. R, et al. 2007. Journal of virology 81 :5413-5417; Moore, P. L., et al. 2008.
  • Residues between 160 and 172 were recently demonstrated to be associated with virus escape to autologous antibody response (Doria-Rose, N. A., et al. 2014. Nature 509:55-62). Although alterations in the variable loop length and glycan content may contribute to resistance to neutralizing antibodies (Doria-Rose, N. A., et al. 2012. Journal of virology 86:8319-8323; Ringe, R., et al. 2012. Virology 426:34-41 ; and van Gils, M. J., et al. 2011.
  • Plasmids, viruses, antibodies, proteins and cells Plasmids encoding HIV-1 envelopes were obtained from different sources as shown in Table S I. Monoclonal antibodies used in the study and TZM-bl cells were procured from the NIH AIDS Research Reagents Reference program and from the IAVI Neutralizing Antibody Consortium (NAC). 293T cells were purchased from the American Type Culture Collection (ATCC). Plasmid DNA encoding BG505-SOSIP.664-D7324, its purified cleaved trimeric protein (Ringe, R. P., et al.
  • Retrovirology 11 was kindly provided by Prof John Moore, Weill Cornell Medical College, New York. Purified gpl20 TripleMut core protein (Feng, Y., et al. 2012. J Biol Chem 287:5673-5686) was obtained from Prof Richard Wyatt, The Scripps Research Institute through the NAC. HIV -2 7312A and its chimeric constructs were provided by Prof Lynn Morris, NICD, Africa.
  • the purified monomeric gpl20 protein was extensively dialyzed with PBS (pH 7.4), concentrated using Amicon® Ultracentrifugal filers (Millipore Inc.) with a 30KDa cut off and stored in -80°C until further use (Figure S4).
  • the trimeric BG505-SOSIP.664 protein was purified using 293F cells essentially as described by Sanders et al (PLoS pathogens 9:el003618). Briefly, the 293F cells were transfected with plasmid encoding DNA encoding both BG505-SOSIP.664 gpl40 envelope and furin (Chung, N. P., et al. 2014. Retrovirology 11 :33). Supernatant containing soluble BG505-SOSIP.664 gpl40 was harvested 72 to 96 hours post transfection, filtered and passed through a lectin agarose column obtained from Galanthus nivalis (Sigma Inc.).
  • BG505-SOSIP.664 was further purified by Sephadex G-200 size exclusion chromatography (AKTA, GE). Trimeric protein fractions were collected, pooled, quality assessed by running in blue native polyacrylamide gel electrophoresis (BN-PAGE) and favorably assessed for their ability to bind to only neutralizing and not to non-neutralizing and MPER directed monoclonal antibodies as described elsewhere (Ringe, R. P., et al. 2013. PNAS 110: 18256-18261) by ELISA.
  • G37080 plasma was diluted to 1 :50 in DMEM containing 10% Fetal Bovine Sera (FBS) and 500 ⁇ of diluted plasma was incubated with 20 ⁇ 1 of beads at room temperature for 45 minutes. Unbound plasma antibodies were separated from ones bound to protein coated beads using a DynaMagTM 15 magnet as described herein. This step was repeated 4-5 times for depletion of plasma antibodies by monomeric gpl20 and 10-12 times in case of BG505-SOSIP.664 coated beads. As a negative control, G37080 plasma antibodies were depleted with uncoated beads in parallel.
  • FBS Fetal Bovine Sera
  • gpl20 and gpl40 ELISA were coated with ⁇ of monomeric 4-2.J41 gpl20 ( ⁇ g/ml) in binding buffer comprising 0.1 M NaHCC (pH 8.6) and incubated overnight at 4° C.
  • gpl20 bound plates were washed once with IX PBS (pH 7.4) and blocked with 5% non-fat milk for 90 min at 37°C.
  • the plates were then washed three times with IX PBS, followed by addition of ⁇ of MAbs as well as the depleted and undepleted plasma antibodies at different dilutions and incubated for lhr at RT.
  • the plates were washed four times with PBS containing 0.1% Tween 20 (PBST) followed by addition of ⁇ of 1 :3000 diluted HRP- conjugated anti-human IgG (Jackson Immunoresearch, Inc.) and further incubated for 45 min at room temperature. Unbound conjugates were removed by washing with PBST and color developed by addition of ⁇ of 3, 3', 5, 5'-tetramethylbenzidine (TMB) (Life Technologies, Inc.) substrate was added. Absorbance was measured at 450 nm in a spectrophotometer.
  • TMB 3, 3', 5, 5'-tetramethylbenzidine
  • Binding of antibodies to BG505-SOSIP.664-D7324 trimeric protein was assessed essentially as described by Sanders et al. in a sandwich ELISA. Briefly, high binding microtiter plates (Nunc, Inc.) was first coated with D7324 antibody at 10ug/ml (Aalto Bio reagents, Dublin, Ireland) followed blocking extra unbound sites with 5% non-fat milk for 90 min at 37°C. 100 ⁇ of BG505.664-D7324 trimeric protein (300ng/ml) and incubated for 45 mins at room temperature. The extent of binding of G37080 plasma antibodies compared to known neutralizing monoclonal antibodies were assessed by addition of primary and HRP- conjugated secondary anti-human antibody as described above.
  • Neutralization assay Neutralization assays were carried out using TZM-bl cells as described herein. Env-pseudotyped viruses were incubated with varying dilutions of depleted plasma antibodies and incubated for an hour at 37 C in a CO? incubator and subsequently 1 X 10 4 TZM-bl cells were added into the mixture in presence of 25 ⁇ g/ml DEAE-dextran (Sigma, Inc.). The plates were further incubated for 48 hours and the degree of virus neutralization was assessed by measuring relative luminescence units (RLU) in a Luminometer (Victor X2, PerkinElmer Inc.).
  • RLU relative luminescence units
  • Pseudotyped viruses were prepared by co-transfection of envelope expressing plasmid with env-deleted HIV-1 backbone plasmid (pSG3AEnv) into 293T cells in 6-well tissue culture plates using FuGENE6 Transfection Reagent (Promega Inc). Cell supernatants containing pseudotyped viruses were harvested 48 hours post-transfection and then stored at -80°C until further use.
  • the infectivity assays were done in TZM-bl cells (1 X 10 5 cells/ml) containing DEAE- Dextran (25 ⁇ g/ml) in 96-well microtiter plates and infectivity titers were determined by measuring luciferase activity using Britelite luciferase substrate (Perkin Elmer) with a Victor X2 Luminometer (Perkin Elmer Inc.).
  • VENS S AEEEIFRPGGGDMRDNWR SEL YK YK V VEIKPL GI APT A AKRR WEREKR A VGL G A VFL G

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Abstract

La recherche en matière de vaccins contre le VIH-1 se concentre principalement sur la conception d'un immunogène pouvant déclencher la production d'anticorps de neutralisation de toutes les variantes (bcnAbs) puissants et à large spectre contre les glycoprotéines d'enveloppe (Env). Les Env de la variante C selon l'invention sont isolés à partir d'un neutraliseur d'élite.
PCT/US2016/039936 2015-07-07 2016-06-29 Glycoprotéines d'enveloppe de variante c du vih-1 WO2017007646A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001060838A2 (fr) * 2000-02-18 2001-08-23 University Of Washington Virus et vaccins ancestraux du sida
WO2005028625A2 (fr) * 2003-09-17 2005-03-31 Duke University Immunogenes consensus/ancestraux
US20050137387A1 (en) * 2000-02-18 2005-06-23 University Of Washington Office Of Technology Licensing Ancestral and COT viral sequences, proteins and immunogenic compositions
WO2016065252A2 (fr) * 2014-10-24 2016-04-28 International Aids Vaccine Initiative Conception d'un immunogène env trimère natif

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001060838A2 (fr) * 2000-02-18 2001-08-23 University Of Washington Virus et vaccins ancestraux du sida
US20050137387A1 (en) * 2000-02-18 2005-06-23 University Of Washington Office Of Technology Licensing Ancestral and COT viral sequences, proteins and immunogenic compositions
WO2005028625A2 (fr) * 2003-09-17 2005-03-31 Duke University Immunogenes consensus/ancestraux
WO2016065252A2 (fr) * 2014-10-24 2016-04-28 International Aids Vaccine Initiative Conception d'un immunogène env trimère natif

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